Difference between revisions of "Part:BBa K1795024"
(→‎iGEM2022_Nanjing-China Experiment)
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==iGEM2022_Nanjing-China Experiment==
 
==iGEM2022_Nanjing-China Experiment==
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[[Image:Nanjing-China-silver-CusS-RFP.jpg|400px|thumb|right|Fluorescence detection of transformed E.coli. at different concentrations of silver ions]]
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[[Image:Nanjing-China-recombination-device-Atox1.jpeg|400px|thumb|right|Homologous recombination device design]]
 
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<b>Group: Nanjing-China 2022</b>
 
<b>Group: Nanjing-China 2022</b>
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We add new documentation to this existing Part, pCusC mKate2 (BBa_K1980007), and expand its applications in silver ions indicator through paper reading and our experiment results.  
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We believe that there are two modes to improve part. One is to optimize the performance of the existing functions of part, and the other is to expand its application scenarios. Here we chose the latter to implement the improvement of BBa_K1795024.
 
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This part consists of the promoter pCusC (BBa_K1980004), a copper responsive promoter activated by the CusS/R two-component system, with a downstream RFP variant (mKate). According to the reference we found, the histidine kinase CusS of the CusR/S two-component system functions as an Ag(I)/Cu(I)-responsive sensor kinase, which is not unexpected due to the similar chemical properties of silver and copper. These findings suggest a model for activation of the histidine kinase through metal binding events in the periplasmic sensor domain. And the histidine kinase of CusS is the key to triggering downstream reactions including RFP expression.  
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The existing part BBa_K1795024 provides Kanamycin resistance to the cell under the Promoter R0010. In the absence of LacI protein and CAP protein, this part promotes KanR transcription. In the presence of LacI protein and CAP protein, this part inhibits KanR transcription.
 
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We also carried out the experimental study including fluorescence detection of edited E. coli at different concentrations of silver ions. The experimental results support our speculation to some extent and further experiments are needed to confirm that.
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Combined with our own project requirements, we hope to engineer it into a reporter gene of homologous recombination. We kept the KanR coding region and removed its initiation codon so that KanR shares the same initiation codon with BpfA. Then Kanamycin resistance can be used as the basis to judge whether the strain is recombinant that displays silver-binding protein. The recombinant is able to grow on the plate with kanamycin, only if the goal sequence and KanR are recombined to the C-terminal of BpfA successfully.
 
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Revision as of 07:14, 12 October 2022


KanR Operon under R0010

Provides Kanamycin Resistance to the cell under the Promoter R0010.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


iGEM2022_Nanjing-China Experiment

Homologous recombination device design

Group: Nanjing-China 2022
Author: Jiankai Liu

We believe that there are two modes to improve part. One is to optimize the performance of the existing functions of part, and the other is to expand its application scenarios. Here we chose the latter to implement the improvement of BBa_K1795024.

The existing part BBa_K1795024 provides Kanamycin resistance to the cell under the Promoter R0010. In the absence of LacI protein and CAP protein, this part promotes KanR transcription. In the presence of LacI protein and CAP protein, this part inhibits KanR transcription.

Combined with our own project requirements, we hope to engineer it into a reporter gene of homologous recombination. We kept the KanR coding region and removed its initiation codon so that KanR shares the same initiation codon with BpfA. Then Kanamycin resistance can be used as the basis to judge whether the strain is recombinant that displays silver-binding protein. The recombinant is able to grow on the plate with kanamycin, only if the goal sequence and KanR are recombined to the C-terminal of BpfA successfully.